Evolutionary Relationships in the Podalyrieae and Liparieae (Fabaceae) Based on Morphological, Cytological, and Chemical Evidence

Plant P1. Syst. Evol. 209:1-31 (1998) Systematics and Evolution © Springer-Verlag 1998 Printed in Austria

Evolutionary relationships in the Podalyrieae and Liparieae (Fabaceae) based on morphological, cytological, and chemical evidence

ANNE LISE SCHUTTE and B.-E. VAN WYK

Received May 21, 1996; in revised version December 11, 1996

Key words: Fabaceae, Podalyrieae, Liparieae. - Morphology, chromosome numbers, chemical compounds, taxonomy, genetic relationships. Abstract: Taxonomic relationships amongst the genera of the southern African tribes Podalyrieae and Liparieae are discussed. Data gained from morphological, cytological and chemical investigations are analyzed cladisfically to determine relationships. The genus Cadia (tribe Sophoreae) is included in the investigation to establish whether it should be transferred to the Podalyrieae. The results clearly indicate that the Podalyrieae and Liparieae are monophyletic and that they should be united, but that Hypocalyptus and Cadia should be excluded. Within the monophyletic group, there are two distinct subcladeg each supported by three apomorphies. The results also show that there is a strong sister relationship between Amphithalea and CoeIidium. In the taxonomic treatment the Liparieae are placed into synonymy under the Podalyrieae and two subtribes are recognized. A key to the genera in the tribe is given, followed by a synopsis of the genera.

When POLHILL (1976, 1981a, c-f) proposed a biogeographic classification for the Podalyrieae and Genisteae sensu BENTHAM (1837, 1839), he divorced the African Podalyrieae, Liparieae and Crotalarieae from the northern temperate Thermo- psideae and Genisteae, and the Australian Mirbelieae and Bossiaeeae. He placed the genera Cyclopia VENT., Podalyria LAM. and Virgilia POIR. in the Podalyrieae on account of the more or less free stamens. Amphithalea Ec~. & ZEYH., Coelidium VOGEL ex WALP., Hypocalyptus THtrNB., Liparia L. and Priestleya DC. were allocated to the Liparieae, based on the fused stamens. POLHILL (1981d, e), however, surmised that it might be sensible to amalgamate the two tribes, but added that a detailed study is needed to clarify the uncertainty regarding relationships between them. Over the last four years, the taxonomy of the Podalyrieae and Liparieae has been the subject of intensive research (SCHUTTE 1995). A series of studies was conducted in which the morphological variation, chromosome numbers and chemical constituents in each of the genera were scrutinized to determine generic delimitations. Some fascinating results were obtained, which emanated in several 2 A.L. SCHUTr~ & B.-E. VAN WYK:

substantial changes at generic level: (1) it was established that Priestleya is paraphyletic, resulting in Priestleya sect. Aneisothea being raised to generic level under the reinstated name Xiphotheca Ec~. & ZEYI~. (ScHtn'~ & VAN WYK 1993); (2) Priestleya sect. Priestleya was found to be congeneric with Liparia and included therein (ScHua'TE & VAN WYK 1994); (3) Podalyria also proved to be paraphyletic, which led to the description of a new genus, Stirtonanthus B.-E. VAN WY~: & A. L. ScritJrrE (VAN WYI,: & SCrlUra'E 1994, 1995a); (4) the genus Calpurnia E. MEY. (tribe Sophoreae) was found to be a sister group of Virgilia and subsequently transferred to the Podalyrieae (VAN WYK & Scrltn'a'E 1995b). An earlier preliminary survey of phylogenetic relationships in the tribes Podalyrieae, Liparieae and Crotalarieae (VAN WYI,: & Sclqm'a'E 1995b), indicated that both the Podalyrieae and Liparieae are monophyletic taxa, which originated from a common ancestor. Hypocalyptus, however, dropped to a basal position, thereby reflecting its incongruous taxonomic position. More information has since then come to light. In the tribe Sophoreae, recent anatomical, chemical and DNA studies have indicated that the genus Cadia FORSSI,:. deviates from other members of the Cadia group (PoIJqmL 1981b) in various characters, e.g. wood anatomy (GASSON 1994), alkaloids (VAt< WYK & al. 1993), chloroplast DNA (DOYLE 1987, 1995). POLnmL (1994) subsequently abandoned the Cadia group and transferred Cadia to the Sophora group. He, however, reiterated the suggestion by VAN WYK & al. (1993), that the genus should possibly be transferred to the Podalyrieae. The objectives of this paper are (1) to discuss the morphological, cytological and chemical variation in the Podalyrieae and Liparieae, (2) to re-examine evolutionary affinities amongst the genera, (3) to provide a predictive classification for the tribes, based on sound empirical evidence, and (4) to investigate the possible inclusion of Cadia in the Podalyrieae.

Morphological characters Growth form. In the Podalyrieae and Liparieae, all the species are long-lived perennials. There is a considerable variation in growth form - from tall upright trees with a main trunk, to erect woody shrubs, to virgate, multi-stemmed shrubs, to small rounded subshrubs or sprawling shrublets. The general trend of a tree-like versus a shrub-like growth form is thus applicable as an evolutionary character. Adaptations to survive recurrent fires have, to a large extent, influenced the life forms of the taxa. The two fire-survival strategies, sprouters (able to resprout after fire) and non-sprouters (obligate reseeders after fire), are important taxonomic characters, particularly at the Specific level (ScmrrwE & al. 1995; Sclatrra~ 1997c, d). At generic level, however, there are no clear-cut discontinuities and these characters appear to have evolved independently in the different genera. Leaves. Leaves are imparipinnate in Virgilia and Calpurnia, digitately trifoliolate in Hypocalyptus and Cyclopia and simple in Amphithalea, Coelidium, Liparia, Podalyria, Stirtonanthus and Xiphotheca (Fig. 1). Petioles are invariably presen t in most genera, except in Amphithalea and Coelidium, where they are strongly reduced or absent and Liparia, where they are totally lacking. Cyclopia and Liparia have decurrent leaf bases and the leaves tend to turn black when dried. Relationships in the Podalyrieae and Liparieae 3

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Liparia has leaves with three or more primary veins arising from the base of the lamina, in contrast to the pinnately veined leaves of the other genera. Stipules are invariably present in all the genera, albeit strongly reduced in size in Amphithalea, Coelidium and Xiphotheca. It seems as if specialization in leaves occurred along the sequence of reduction as proposed by DOe,MZR (1945, 1946) and Pou-ImL (1981a): compound to simple; petioles present to absent; stipules conspicuous to strongly reduced. Infloreseenees. According to POLHILL (1976), inflorescences tend to be rather labile in the Papilionoideae. In the Podalyrieae and Liparieae inflorescence structure is a useful character at both inter- and infrageneric level. Terminal, many- flowered racemes occur in Hypocalyptus (Fig. 2). Virgilia and Calpurnia have subterminal and axillary racemes or panicles. The rest of the Podalyrieae and Liparieae have axillary, simple racemose inflorescences, with modifications in the number of flowers, length of the peduncle and length of the inflorescence axis (Fig. 2). In Liparia the flowers are borne on lateral short shoots, terminating in a small apical extension of the inflorescence axis. These brachyblasts are either contracted

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into many-flowered heads subtended by petaloid, sterile bracts, or few-flowered units in which a decrease in the length of the axis eventually leads to decussate two- or four-flowered inflorescences (Scmn:~ & VAN WYK 1994). Xiphotheca, Coelidium and several species of Amphithalea invariably have geminate (paired) flowers. The other species of Amphithalea are either bifloral at the base of the flowering branch, becoming unifloral towards the apex, or uniformly single- flowered. Specialization appears to culminate in Cyclopia, where the inflorescences are consistently unifloral and supported by two bracts, very rarely three or four bracts (ScIatrrTE, pers. obs.), with the flower situated in the axil of the uppermost bract. The bracts are generally sheathing (i.e. with a broad point of attachment) in the Podalyrieae and Liparieae (Fig. 3). Tridentate bracts, possibly due to an extreme reduction in leaf size and subsequent fusion with the adjacent stipules, have been observed in Hypocalyptus, Virgilia, Stirtonanthus and Podalyria. The bracts are caducous at an early stage in Podalyria, Virgilia and Calpurnia. Xiphotheca and three species of Coelidium have the bracts fused with the pedicel at the base. Most genera lack bracteoles, except Hypocalyptus, Xiphotheca, Virgilia and Calpurnia, where they are either conspicuous (Hypocalyptus, Xiphotheca phylicoides A. L. SCrIUTTE & B.-E. VAN WYK, X. canescens (Tmn~.) A. L. ScI-IUTTz & B.-E. VAN WYK, X. elliptica (DC.) A. L. Scmn:TE & B.-E. VAN WYK, or strongly reduced (Virgilia oroboides ADAMSON, Calpurnia and the remainder of Xiphotheca). The structure and evolutionary tendencies of inflorescences in the Fabaceae have been discussed by WE~Er~IN6 (1989). Trends evident in the Podalyrieae and Liparieae include the following: inflorescence position terminal to axillary; inflorescence type racemose to geminate; inflorescences extended to congested; many-flowered to few-flowered to unifloral inflorescences; terminal extension of inflorescence axis absent to present; bracts persistent to caducous; bracteoles present to strongly reduced or absent. Flowers. Calyx. In the Podalyrieae and Liparieae, the upper two calyx lobes are invariably fused higher up than the lower three lobes (Fig. 4) This character breaks down only in three species of Calpurnia, where the sinus between the upper two lobes is subequal to the others. According to POLIqmL (1976), the Podalyrieae and Liparieae calyx type is the most ubiquitous and apparently basic form in the subfamily. However, some specialization is found in the carinal calyx lobe in Cyclopia and especially Liparia, where it is longer or larger than the upper four and often distinctly keeled. Virgilia has a two-lipped calyx, with the lower three lobes fused into a trifid lower lip. An intrusive calyx base is characteristic of all the genera, except Xiphotheca, Amphithalea, Coelidium and a few species of Calpurnia (Fig. 3). These taxa have attenuate calyx bases, with the receptacular part (hypanthium) often prominent. There is, however, one species of Xiphotheca which has an intrusive calyx base (ScI~tn'TE & VAN WYK 1993, Sci-Itrr~ 1997a), but it is here regarded as a secondary development. Corolla. There are some noteworthy patterns of divergence in the corolla of the Podalyrieae and Liparieae (Fig. 5). Apart from Amphithalea, Coelidium and Xiphotheca, all the genera have firmly textured flowers, adapted to pollination by large xylocopid bees and presumably also birds and small non-flying mammals (REBELO 1987, SCHm'TE & VAN WYK 1994). The flowers are yellow in all species of 6 A.L. SCHUTTE& B.-E. VAN WVK:

Fig. 3. Flowers of the Podalyrieae and Liparieae in lateral view (note the structure of the calyx, position of the bracts and presence or absence of bracteoles). A Hypocalyptus coluteoides; B Amphithalea micrantha; C Coelidium tortile; D Calpurnia intrusa; E Cyclopia intermedia; F Stirtonanthus taylorianus; G Liparia umbellifera; H Xiphotheca phylicoides; I Virgilia divaricata; J Liparia splendens subsp, splendens; K Podalyria argentea. - Scale in mm

Calpurnia, Stirtonanthus, Cyclopia, Xiphotheca and most species of Liparia (one species has bright orange-red flowers and two others have a lemon-yellow flower colour). White, pink or purple flowers, often with a white or dark violet nectar guide, predominate in Virgilia Podalyria, Coelidium and Amphithalea. Hypoca- lyptus is unusual in having magenta pink flowers with a yellow nectar guide (see discussion on anthocyanins below). The standard petal is large, thickly textured, usually strongly reflexed, with callosities at the base and emarginate at the apex in Liparia, Hypocalyptus and members of the Podalyrieae (Fig. 5). Keel petals are rostrate or beaked and lack a distinct pocket or auricle. Not even the wing petals are pocketed or auriculate, but Relationships in the Podalyrieae and Liparieae 7

H Fig. 4. Calyces of the Podalyrieae and Liparieae (opened out with the upper lobes to the left). A Stirtonanthus chrysanthus; B Liparia angustifolia; C Xiphotheca elliptica; D Amphithalea ericifolia; E Virgilia divaricata; F Hypocalyptus coluteoides; G Coelidium tortile; H Podalyria myrtillifolia; I Calpurnia intrusa; J Cyclopia intermedia. - Scale in mm

petal sculpturing is sometimes present. Cyclopia is exceptional in having distinct grooves on the standard petal, which probably act as nectar guides and well- developed pockets on the keel and wing petals (Fig. 5). Amphithalea, Coelidium and Xiphotheca have relatively unspecialized corollas with the keel petals obtuse and distinctly pocketed and auriculate (Fig. 5). In these genera the pocket on the wing petal is a thickened lobe, formed on the inside (abaxial side). Wing petal sculpturing is invariably present in the upper basal area. Stamens. Traditionally the fusion of the stamens has been used to discern the Liparieae from the Podalyrieae (PoI~nlLL 1976, 1981d, e). In the Podalyrieae the stamens are free or almost free to the base, but in the Liparieae the degree of fusion varies from diadelphous in Liparia, Xiphotheca and Amphithalea to monadelphous in Coelidium, to a closed tube in Hypocalyptus (Fig. 6). In fact, the generic circumscription of Coelidium is based solely on this character. Both Amphithalea and Coelidium have a well-developed hypanthium, which implies that the stamens are fused into an open sheath for at least that part. This means that the only real difference between the two genera lies in the degree of fusion of the vexillary stamen (see variation in Fig. 6). The stamens are thickened at the base in the Podalyrieae and the vexillary filaments are modified to form one or two nectar wells in Cyclopia, Calpurnia, A. L. SC~tn'TE & B.-E. VAN W¥I(:

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Podalyria, Virgilia, Liparia and Stirtonanthus. Hairy stamens occur in Virgilia (Fig. 6) and one species of Calpurnia. Dimorphic anthers seem to be associated with a beaked keel, those of the unspecialized flowers being less differentiated (Fig. 6). The anthers are alternately short and dorsifixed or long and basifixed or subbasifixed throughout the genera. Pistils. Hypocalyptus deviates from the other genera in having the pistil stipitate. All genera have the style curved upwards and in Podalyria, Stirtonanthus, Amphithalea and CoeIidium the base of the style is often hairy. The number of ovules varies from one or two in Coelidium (Gl~_~3Y 1980) and Amphithalea (GRANBY 1985), to up to thirty in Hypocalyptus (DAHLGREN 1972). These dif- ferences are of use only at infrageneric level. Fruits. The significance of fruit characters (i.e. of the pods and seeds) in the taxonomy of the Fabaceae is well known (CORNER 1951, 1976; DAHLGREN 1975; POLHICL 1976, 1981a; ESAU 1977; Dt~m 1981; GVNN 1981a, 1981b; PATE & Kuo 1981; MAYNIN~& VAN STADEN 1987). However, POLHI~L (1976) pointed out that the weighting of fruit characters at generic and tribal level has, in some instances, led to artificial segregation of otherwise natural groups. The variation in fruit structure in the Podalyrieae and Liparieae is illustrated in Fig. 7. Pods are laterally compressed and constricted between the seeds in Xiphotheca, Virgilia, Calpurnia and one species of Hypocalyptus, but this character seems to have evolved independently in the different genera. The remaining taxa have inflated pods. Apart from Hypocalyptus, which has tardily dehiscent pods

Fig. 7. Fruits of the Podalyrieae and Liparieae in lateral view. A Liparia striata; B Xiphotheca guthriei; C Amphithalea ericifolia; D Coelidium vlokii; E Cyclopia genistoides; F Stirtonanthus taylorianus; G Calpurnia intrusa; H Hypocalyptus coluteoides; I Virgilia divaricata; J Podalyria rnyrtillifolia. Scale in mm Relationships in the Podalyrieae and Liparieae 11

(ScmrrTE, pers. obs.), seeds are released by the explosive mechanism (POLHmC 1976) in all the other genera. Both tribes have sessile, obliquely oblong to ovate pods, except in Hypocalyptus, where the pods are distinctly stipitate and where the three species each have a characteristic pod shape. In most genera, the pods are many-seeded, but in Amphithalea and Coelidium the number is reduced to one or rarely two seeds per pod. Evolutionary trends in fruit characters have been discussed by DUDIK (1981) and POLHILL (1981a). The following trends apply in the Podalyrieae and Liparieae: legume many-seeded to one- or two-seeded; legume dehiscent to tardily dehiscent; and seed with an aril to seed without an aril. Seeds. Characters of the seed have traditionally been regarded as conservative (MANNIN~ & VAN STADEN 1987, VAN STADEN & al. 1989). According to POLHILL (1976) aril characters have previously been overweighted at the generic level, but neglected at the generic group or tribal level in the Genisteae sensu BENTHAM. In the South African Papilionoideae, the Podalyrieae and Liparieae represent the arillate tribes. They differ from the Australian tribes in having a collar-like aril, not hooded, channelled, papillate or with a rugose lobe distal to the radicular lobe as in the Bossiaeeae and Mirbelieae (CRISP & WESTON 1987). The aril is reduced to a non-fleshy rim aril in Virgilia, Stirtonanthus and Calpurnia, but thick and fleshy in the other taxa. Further specialization is found in Coelidium and Amphithalea, where the aril is prolonged towards the lens area (see Fig. 9 in VAN WYK & SCHUTTE 1995b). Hypocalyptus is anomalous in having the aril continuous around the hilum, whilst all the other genera have it interrupted at the radicular end of the hilum. Stirtonanthus is unusual in the oblique orientation of the lens alignment (VAN WYI,: & ScHtrrrz 1994). As shown by MANNINC & VAN STADEN (1987), the shape and position of the micropyle are of taxonomic importance in subfam. Papilionoideae. In the Podalyrieae and Liparieae, Hypocalyptus deviates in having an ypsaloid micropyle, which is situated outside the hilar region. The remaining genera all have a punctate micropyle situated within the hilar region (see Fig. 9 in VAN WYK & SCHUTT~ 1995b). Secondary xylem. BARETTA-KUIPERS (1981) has shown that the structure of secondary wood is taxonomically useful in the Fabaceae at subfamily and tribal levels. She, however, omitted shrubby legumes from her study, as the xylem in these do not have all the characters of the xylem of trees (mature wood). Since most of the genera of the Podalyrieae and Liparieae include at least some woody species, which can become 15 to 30 years old, a study of the secondary xylem of these taxa was undertaken. Two main groups are apparent: those with the vessels arranged in small, isolated, radial or tangential groups as in Virgilia, Calpurnia and Hypocalyptus, and those with the vessels arranged in large confluent (dendritic) groups as in Podalyria, Stirtonanthus, Cyclopia and all the genera of the Liparieae, except Hypocalyptus (VAN WYK & SCHUTTE 1995b). The isolated type of arrangement is found in the supposedly more primitive tribes of the Papilionoideae (METCALFE& CHALK 1950, BARETTA-KUIPERS1981), which seems to indicate that the Confluent type of arrangement is more advanced. According to BAAS • SCHWEINGRUBER(1987), FuJII & al. (1994) and BAAS (pets. comm.) the diffuse arrangement of vessels is found in species occurring in tropical regions, 12 A.L. SClJUTa'E & B.-E. VAn WYI(: whilst the more complex dendritic patterns are associated with species found in strongly seasonal habitats, e.g. the Cape fynbos region or other Mediterranean ecosystems. This trend has been explicitly documented by CARLQUIST(1987), BAAS & SCnWEn~GRtn~ER(1987), BAAS & al. (1988) and WHEELER & BAAS (1991). Antipodals. Antipodals in the female gametophyte have proved to be of taxonomic value at the tribal level in the Australian Papilionoideae (CAMERON & PRAKASH 1990). Suggestions that the Australian Bossiaeeae and Mirbelieae may be closely related to the Podalyrieae and Liparieae (CRise & WESTON 1987), urged an investigation into the structure of antipodals in these two tribes, especially in view of the aberrant position of Hypocalyptus in the Liparieae. A detailed discussion of the results will be published elsewhere (ScI-IVTTE 1997b). It was found that the antipodals are prominent and persistent at least until anthesis in Liparia, Xiphotheca, Amphithalea, Coelidium and the genera of the Podalyrieae. Hypocalyptus, however, has inconspicuous and ephemeral antipodal cells. Thus, unlike the Bossiaeeae and Mirbelieae, antipodals are neither gigantic nor totally absent in the Podalyrieae and Liparieae. A direct affinity between the South African and Australian tribes, based on this character, seems unlikely.

Cytological characters Chromosome counts are known for all the genera of the Podalyrieae and Liparieae. With the exception of Cyclopia, Virgilia and Hypocalyptus, all have 2n = 18 (DAttLGREN 1967; GRANBY 1980; GOLDBLATT 1981a; POLIJmL 1981d, e; SCHUTTE 1995). Cyclopia and Virgilia are the only known polyploids, the latter a hexaploid with 2n = 54 (VAN WYI( 1986, GOLDBLAra" 1981b) and the former, with an apparent polyploid range of 2n = 36 (GOLDBLATT 1981b), 54 and 4-126 (ScHuTTE 1995). A basic chromosome number of x = 9 seems most likely for the two tribes, as was suggested by GOLDBLATT (1981a). Hypocalyptus, however, is atypical in having 2n = 20, and therefore a base number of x= 10 (GOLDBLATT 1981b, VAN WYK & SCmJTTE 1995b).

Chemical characters Alkaloids. Virtually nothing was known about the distribution of alkaloids in the tribes Podalyrieae and Liparieae prior to our studies. Only .Virgilia (MEARS & MABRY 1971, VAN EIJK & RADEMA 1982, VAN EIJK & al. 1982) and some species of Calpurnia (RADEMA & al. 1979; ASRES & al. 1986a, b) had been studied earlier and were known to contain unique combinations of quinolizidine alkaloids. The distribution of the major alkaloids found in the genera of the Podalyrieae and Liparieae are summarized in Table 1. Hypocalyptus and Cyclopia are not included, since no alkaloids could be detected in them. Each of the other genera appears to have a characteristic combination of major alkaloids. The marked decrease in the number of compounds found in the genera of the Liparieae is noteworthy. No less than 16 different alkaloids were found in Podalyria and Stirtonanthus (VAN WYK & al. 1992). Most species of Podalyria produce tetracyclic quinolizidine alkaloids such as sparteine, lupanine, isolupanine, 13a-hydroxylupanine and Relationships in the Podalyrieae and Liparieae 13

Table 1. The distribution of major alkaloids in the genera of the Podalyrieae and Liparieae. *** present in all species examined, ** present in most species examined, * present only in a few species examined, ÷ present in trace quantities only. CALP Calpurnia, VIRG Virgilia, PODA Podalyria, STIR Stirtonanthus, LIPA Liparia, XIPH Xiphotheca, AMPH Amphi- thalea, COEL Coelidium

Alkaloids CALP VIRG PODA STIR LIPA XIPH AMPHCOEL

Anabasine *** Ammodendrine ÷ * Lupinine * *** *** Epilupinine * *** 4/3-O-(T-pyrrolyl-carboxyl)-epilupinine * 4fl-hydroxy- 11-O-(U- pyrrolyl-carboxyl)-epilupinine *** Lusitanine * Sparteine ** ** + *** ** a-isosparteine + ** ** 11,12-dehydrosparteine ** Lupanine ** *** + ** Isolupanine ** * ** 17-oxolupanine ** 3-hydroxylupanine * 13a-hydroxylupanine *** ** ** *** ** 4fl, 13c~-dihydroxylupanine * 3, 13-dihydroxylupanine * 10, 13-dihydroxylupanine * 3, 4, 13-trihydroxylupanine * Digittine * Lebeckianine * Calpurmenine ** Lupanine 13a-angelate * * * Lupanine 13a-tigliate ** Cajanifoline ** Sessilifoline * Pearsonine * Oroboidine *** ** *** Calpaurine * 13o~-pyrrolyl-carboxyl -acid-ester ** Aphylline * Virgiline *** *** ** Virgiline-pyrrolyl- carboxyl-acid-ester *** *** *** * Virgiboidine *** Dihydrovirgiboidine ** Virgilidone ** Dihydrovirgilidone * 14 A.L. SCHUTTE• B.-E. VAN WYK: cajanifoline (an ester). Some species also contain other esters (pearsonine, sessilifoline, lupanine 13a-angelate), hydroxylated lupanines and aphylline. The three species of Stirtonanthus, however, were found to accumulate virgiline, 13a- hydroxylupanine and two totally different esters, oroboidine and virgiline-pyrrolyl- carboxylic acid ester. These two different sets of alkaloids in the two taxa, supported other deviations in morphological characters (VAN WYK • SCHUTTE 1994, 1995a) and hence, proved that they are dissimilar. In fact, the compounds contained in Stirtonanthus show more resemblance with Virgilia than with Podalyria. In a detailed study of the alkaloids of Virgilia, a total of 44 alkaloids were detected; 43 of the quinolizidine type and one bipiperidyl alkaloid (GRZINWALD& al. 1989, VEEN & al. 1991). Virgilia is exceptional in containing also tricyclic quinolizidine alkaloids in addition to bi- and tetracyclic compounds. The presence of virgiline and its carboxylic acid ester as major compounds in Calpurnia, Virgilia and Stirtonanthus, suggests a close relationship between these genera and supports the transfer of Calpurnia to the Podalyrieae (VANWYK & SCHUTTE 1995b). Anabasine (a bipiperidyl alkaloid) and lupinine (a bicyclic quinolizidine alkaloid) were detected as major alkaloids in Xiphotheca. A totally different set of alkaloids was found in Liparia (including Priestleya s. str.) where a combination of lupanine, isolupanine, 13ct-hydroxylupanine, sparteine, oL-isosparteine, 11,12- dehydrosparteine (all tetracyclic quinolizidine alkaloids) and minor quantities of ammodendrine (a bipiperidyl alkaloid) were located (VAN WYK & al. 1991a, b). These findings clearly support the recent reinstatement of Xiphotheca (SCHtrrTE & VAN WYI~ 1993), as well as the inclusion of Priestleya s. str. into Liparia (ScHUrTE & VAN WYK 1994). Amphithalea and Coelidium seem to have very few alkaloids. ARNDT & DU PLESSIS (1968) investigated the alkaloids of one species of Coelidium. They recorded ammodendrine as the major compound and isopiperideine and oz-aldotripiperideine as minor alkaloids. A wider survey of the alkaloids of Amphithalea and Coelidium is currently in progress (VAN WYK & al., unpubl.). Ammodendrine has been detected as a major alkaloid in both genera and quinolizidine alkaloids appear to be absent. The concentration of alkaloids present in the species of Coelidium investigated seems to be higher than in the Amphithalea species, but yields are very variable, and many species have almost no detectable alkaloids. The biogenetic pathways along which quinolizidine alkaloids are produced have been discussed by various authors (NOWACKI& WALLER 1977; SALATINO & GOTTLIEB 1980, 1981; GOMEZ & al. 1981). VAN WY~ & VERDOORN(1990) used this information in the Crotalarieae to construct a phylogeny for the tribe. Following these authors, the biosynthetic routes in the Podalyrieae and Liparieae are illustrated diagrammatically in Fig. 8. The various steps involved in the pathways are explained in the references mentioned above and are not repeated here. Five groups are apparent: (1) a group producing bipiperidyl alkaloids as major compounds, e.g. Coelidium, Amphithalea and Xiphotheca (Liparia has ammodendrine as a major compound in one species only); (2) a group with bicyclic quinolizidine alkaloids, e.g. Virgilia, Calpurnia and Xiphotheca; (3) a group accumulating 13oz-hydroxylupanine in all or most species, e.g. Calpurnia, Virgilia, Relationships in the Podalyrieae and Liparieae 15 =Est )F ~Est I (~Vir ~Vo

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Calpurnia Virgilia Stirtonanthus

Fig. 8. Diagrammatic representation of biosynthetic pathways of Podalyrieae and Liparieae alkaloids and a comparison of the routes present in the different genera. Direction of arrows suggests increasing specialization. Biosynthetic pathways: 1 lysine pathway; 2 bipiperidyl pathway; 3 quinolizidine pathway; 4 bicyclic quinolizidine pathway; 5 tetracyclic quinolizidine pathway; 6 10-oxo pathway; 7 tricyclic quinolizidine pathway. Compounds: Am ammodendrine; An anabasine; Li lupinine; Lu lupanine; Sp sparteine; Ap aphylline; Vir virgiline; Vo virgiboidine; OH monohydroxylated alkaloids; PH polyhydroxylated alkaloids; Est esters of mono- and polyhydroxylated alkaloids. Solid lines indicate presence; broken lines indicate absence 16 A.L. SCHUTTE• B.-E. VAN WYK: Relationships in the Podalyrieae and Liparieae

Podalyria, Stirtonanthus and Liparia; (4) a group specializing in the 10-oxo pathway with subsequent esterification, e.g. Stirtonanthus, Virgilia and Calpurnia; (5) a group producing lupanine-type esters, e.g. Calpurnia, Virgilia, Stirtonanthus, Podalyria and Liparia. These different specializations are included as characters in the cladistic analyses below. Canavanine. A detailed survey of the occurrence of canavanine, a non-protein amino acid, in legume seeds was carried out by BELL & al. (1978). Once again, Hypocalyptus is incongruent in being the only member of the Podalyrieae and Liparieae, which does accumulate canavanine in its seeds. All three the species tested positive for canavanine. Elsewhere, this component is known from the tribes Desmodieae, Indigofereae, Trifolieae, Galegeae, Robinieae, Sesbanieae, Bos- siaeeae and some genera of the Mirbelieae, Phaseoleae, Coronilleae, Vicieae and Millettieae. Phylogenetically, the absence of canavanine is here interpreted as apomorphic at the suprageneric level. Anthoeyanins. The anthocyanins in the pink-, purple- or white-flowered species of Virgilia, Podalyria, Coelidium, Amphithalea and Hypocalyptus have been studied by VAN WYK & WI~r~R (1994) and VAN WYK & al. (1995). It was established that flower pigmentation is conservative within the genera but that each of the genera contains an unique combination of anthocyanins. Virgilia and Podalyria differ from each other in the presence of a coumaroyl ester, cyanidin-3-(p-coumaroylglucoside), in Podalyria, whilst acetic acid esters of cyanidin-3-glucoside and peonidin-3-glucoside are restricted to Virgilia (VANWY~C & WINTER 1994). In the Liparieae, the flowers of Amphithalea and Coelidium accumulate esters of cyanidin-3-glucoside, but in Amphithalea it is esterified with acetic acid and in Coelidium with coumaric acid (VAN WYK & al. 1995). The 3-sophorosides of cyanidin and pelargonidin are responsible for the red flower colour in Liparia. All these genera contain cyanidin-3-glucoside, except Hypocalyptus, where malvidin-3-glucoside is the only compound present in the flower petals. This indicates that even in its floral pigments, Hypocalyptus deviates from the rest of the Liparieae. The presence of esters of anthocyanins and cyanidin-3-glucoside in all the pink- and purple-flowered genera (excluding Hypocalyptus) offers additional evidence that the Podalyrieae and Liparieae are monophyletic. In particular, the accumulation of esters of anthocyanins, appears to be unique for the two tribes. All other legumes for which data are available (and Hypocalyptus) have glucosides only.

Cladistic analyses Cladograms were generated by using the computer software package Hennig 86 (FARRIS 1988). Character states were polarized using the method of outgroup comparison. The "mhennig*", "bb*" and "ie" algorithms were applied to produce trees of minimal length. Autapomorphies have been excluded from the analyses as they serve no purpose as grouping characters. Four separate analyses of relationships were carried out (Tables 2-5; Figs. 9, 10). In the first analysis, the Mirbelieae, Crotalarieae and an hypothetical ancestor were used as outgroups, due to the perplexity regarding the affinity of Table 2. Characters and character states used for the cladistic analysis of the genera of the Podalyrieae and Liparieae, with the Mirbelieae, Crotalarieae, Cadia and an hypothetical ancestor as outgroups. The fully resolved cladogram generated from this data set is shown in Fig. 9 Genera Character states Hypothetical Ancestor 00000 00000 00000 00000 00000 O0 Mirbelieae 1 ?000 00000 00001 01100 00000 O0 Crotalarieae 11100 00000 00101 O1100 10000 01 Cadia 00000 00000 00000 000?0 10110 01 Calpurnia 00000 10101 01101 01110 11110 11 Cyclopia 11100 00101 01101 O1110 00000 O1 Podalyria 11100 00101 01101 01110 10010 11 Stirtonanthus 11100 00101 01101 01110 10110 11 Virgilia 00000 10101 01001 O1110 11110 O1 Amphithalea 11111 01110 10111 11111 00001 01 Coelidium 11111 01110 10111 11111 00001 O1 Hypocalyptus 10100 00001 011 O0 00000 00000 O0 Liparia 11110 00101 01101 01110 10011 01 Xiphotheca 11111 01100 10101 01111 01000 01 Characters 1. Habit: trees (0); shrubs (1). 2. Secondary xylem: vessels arranged in small tangential and/or radial groups (0); vessels arranged in large confluent groups (1). 3. Leaf type: pinnate (0); digitate or simple (1). 4. Petiole: present (0); strongly reduced or absent (1). 5. Stipules: present (0); strongly reduced or absent (1). 6. Hairy filaments: absent (0); at least sometimes present (1). 7. Inflorescence type: not geminate (0); geminate (1). 8. Bracteoles: present and conspicuous in most taxa (0); inconspicuous or absent in most taxa (1). 9. Hypanthium: not prominent (0); prominent (1). 10. Calyx base: not intrusive (0); intrusive (1). 11. Wing petals: not lobed towards the inside (0); thickly lobed towards the inside (1). 12. Keel apex: obtuse (0); rostrate or beaked (1). 13. Stamen fusion: totally free (0); fused (1). 14. Ovule number: several ovules (0); 1 or 2 ovules (1). 15. Seed aril: continuous around hilum (0); interrupted at micropylar end (1). 16. Seed aril shape: aril not extended towards the lens (0); aril extended towards the lens (1). 17. Seed micropyle type: ypsaloid (0); punctate (1). 18. Seed micropyle position: outside the hilum (0); inside the hilum or on the rim (1). 19. Antipodals: not persistent (0); persistent (1). [Giant antipodals as in some Mirbelieae (CAMERON & PRAKASH 1990) is considered to be a separate development.] 20. Piperidyl alkaloids: low concentrations or absent (0); present as major compounds in most taxa (1). 21. Tetracyclic quinolizidine alkaloids: absent (0); present (1). 22. Bicyclic quinolizidine alkaloids: absent (0); present (1). 23. Carboxylic acid esters of alkaloids: absent (0); present (1). 24. Esterification of monohydroxylated lupanines: absent (0); present (1). 25. Ammodendrine: absent (0); present as a major compound in at least some taxa (1). 26. Esterification of polyhydroxylated lupanines: absent (0); present (1). 27. Canavanine: present (0); absent (1). Table 3. Characters and character states used for the cladistic analysis of the genera of the Podalyrieae, using the Mirbelieae as an outgroup. The fully resolved cladogram generated from this data set is shown in Fig. 10A Genera Character states Mirbelieae 1 ?000 00000 00000 00000 00000 00000 000 Calpurnia 00000 00000 01001 11000 01010 11110 111 Cyclopia 11100 10111 01011 01011 00000 00100 010 Podalyria 11100 10101 01011 01011 00000 10110 110 Stirtonanthus 11100 10101 01011 01011 01010 11110 110 Virgilia 00000 00101 01011 11011 01010 11110 011 Amphithalea 11111 11001 10100 00100 10101 00101 010 Coelidium 11111 11001 10100 00100 10101 00101 010 Liparia 11110 10111 01011 01111 00000 10111 010 Xiphotheca 11111 11000 00100 00100 00001 00100 011 Characters 1. Habit: trees (0); shrubs (1). 2. Secondary xylem: vessels arranged in small tangential and/or radial groups (0); vessels arranged in large confluent groups (1). 3. Leaf type: pinnate (0); digitate or simple (1). 4. Petiole: present (0); strongly reduced or absent (1). 5. Stipules: present (0); strongly reduced or absent (1). 6. Inflorescence position: not axillary (0); axillary (1). 7. Inflorescence type: racemose (0); geminate (1). 8. Bracts: not sheathing (0); sheathing (1). 9. Sterile bracts: absent (0); present (1). 10. Bracteoles: present (0); absent (1). 11. Hypanthium: not prominent (0); prominent (1). 12. Calyx base: not intrusive (0); at least sometimes intrusive (1). 13. Wing petals: not lobed towards the inside (0); thickly lobed towards the inside (1). 14. Corolla: not firmly textured (0); firmly textured (1). 15. Keel apex: obtuse (0); rostrate or beaked (1). 16. Filaments: not hairy (0); at least sometimes hairy (1). 17. Filaments: not thickened at base (0); thickened at base (1). 18. Stamen fusion: totally free or slightly fused (0); fused high up (1). 19. Nectar well: upper stamens not modified (0); upper stamens modified to form a circular opening (1). 20. Anthers: not dimorphic or slightly dimorphic (0); strongly dimorphic (1). 21. Ovule number: several ovules (0); 1 or 2 ovules (1). 22. Seed aril: fleshy (0); non-fleshy (1). 23. Seed aril shape: aril not extended towards the lens (0); aril extended towards the lens (1). 24. Seed aril type: collar-like (0); rim-like (1). 25. Piperidyl alkaloids: low concentrations or absent (0); present as major compounds in most taxa (1). 26. Tetracyclic quinolizidine alkaloids: absent (0); present (1). 27. Carboxylic acid esters: absent (0); present (1). 28. Canavanine: present (0); absent (1). 29. Esterification of monohydroxylated lupanines: absent (0); present (1). 30. Ammodendrine: absent (0); present as a major compound in at least some taxa (1). 31. Esterification of polyhydroxylated lupanines: absent (0); present (1). 32. Antipodals: ephemeral or absent (0); persistent (1). 33. Bicyclic quinolizidine alkaloids: absent (0); present (1). A. L. SCHUTrE & B.-E. VAN WYK: Relationships in the Podalyrieae and Liparieae 19

Table 4. Characters and character states used for the cladistic analysis of the genera of the Podalyrieae, using the Crotalarieae as an outgroup. The fully resolved cladogram generated from this data set is shown in Fig. 10B Genera Character states Crotalarieae 11100 00000 00000 00000 00000 00000 00 Calpurnia 00000 00000 01001 11100 00011 01111 11 Cyclopia 11100 10111 01011 01110 10101 10010 01 Podalyria 11100 10101 01011 01110 10101 00111 01 Stirtonanthus 11100 10101 01011 01110 00001 01111 01 Virgilia 00000 00101 01011 11110 00011 01110 11 Amphithalea 11111 11001 10100 00001 11100 10000 01 Coelidium 11111 11001 10100 00001 11100 10000 01 Liparia 11110 10111 01011 01010 10101 00100 01 Xiphotheca 11111 11000 00100 00000 10110 10010 11 Characters 1. Habit: trees (0); shrubs (1); 2. Secondary xylem: vessels arranged in small tangential and/or radial groups (0); vessels arranged in large confluent groups (1). 3. Leaf type: pinnate (0); digitate or simple (1). 4. Petiole: present (0); strongly reduced or absent (1). 5. Stipules: present (0); strongly reduced or absent (1). 6. Inflorescence position: not axillary (0); axillary (1). 7. Inflorescence type: racemose (0); geminate (1). 8. Bracts: not sheathing (0); sheathing (1). 9. Sterile bracts: absent (0); present (1). 10. Bracteoles: present (0); absent (1). 11. Hypanthium: not prominent (0); prominent (1). 12. Calyx base: not intrusive (0); intrusive (1). 13. Wing petals: not lobed towards the inside (0); thickly lobed towards the inside (1). 14. Corolla: not firmly textured (0); firmly textured (1). 15. Keel apex: obtuse (0); rostrate or beaked (1). 16. Filaments: not hairy (0); at least sometimes hairy (1). 17. Filaments: not thickened at base (0); thickened at base (1). 18. Stamen fusion: fused high up (0); slightly fused or totally free (1). 19. Nectar well: upper stamens not modified (0) upper stamens modified to form a circular opening (1). 20. Ovule number: several ovules (0); 1 or 2 ovules (1). 21. Seed aril: non-fleshy (0); fleshy (1). 22. Seed aril shape: aril not extended towards the lens (0); aril extended towards the lens (1). 23. Seed aril type: rim-like (0); collar-like (1). 24. Pods: generally inflated (0); compressed (1). 25. Piperidyl alkaloids: present as major compounds in most taxa (0); low concentrations or absent (1). 26. Tetracyclic quinolizidine alkaloids: present (0); absent (1). 27. Carboxylic acid esters: absent (0); present (1). 28. Esterification of monohydroxylated lupanines: absent (0); present (1). 29. Ammodendrine: present as a major compound in at least some taxa (0); absent (1). 30. Esterification of polyhydroxylated lupanines: absent (0); present (1). 31. Bicyclic quinolizidine alkaloids: absent (0); present (1). 32. Antipodals: not persistent (0); persistent (1). 20 A.L. SCHUTTE& B.-E. VAN WYK:

Table 5. Characters and character states used for the cladistic analysis of the genera of the Podalyrieae, using the genus Cadia as outgroup. The fully resolved cladogram generated from this data set is shown in Fig. 10C Genera Character states Cadia 00000 10000 00000 00000 00000 0000 Calpurnia 00000 00000 01001 11100 00011 0000 Cyclopia 11100 10111 01101 10111 01011 0110 Podalyria 11100 10101 01101 10111 01011 0010 Stirtonanthus 11100 10101 01101 10111 00011 0000 Virgilia 00000 00101 01101 11011 00011 0000 Amphithalea 11111 11001 10010 00100 11111 1111 Coelidium 11111 11001 10010 00100 11111 1111 Liparia 11110 10111 01101 10111 01011 0011 Xiphotheca 11111 11000 00010 00100 01011 1110

Characters 1. Habit: trees (0); shrubs (1); 2. Secondary xylem: vessels arranged in small tangential and/or radial groups (0); vessels arranged in large confluent groups (1). 3. Leaf type: pinnate (0); digitate or simple (1). 4. Petiole: present (0); strongly reduced or absent (1). 5. Stipules: present (0); strongly reduced or absent (1). 6. Inflorescence position: terminal (0); axillary (1). 7. Inflorescence type: racemose (0); geminate (1). 8. Bracts: not sheathing (0); sheathing (1). 9. Sterile bracts: absent (0); present (1). 10. Bracteoles: present (0); absent (1). 11. Hypanthium: not prominent (0); prominent (1). 12. Calyx base: not intrusive (0); intrusive (1). 13. Corolla: not firmly textured (0); firmly textured (1). 14. Wing petals: not lobed towards the inside (0); thickly lobed towards the inside (1). 15. Keel apex: obtuse (0); rostrate or beaked (1). 16. Filaments: not thickened at base (0); thickened at base (1). 17. Filaments: not hairy (0); hairy (1). 18. Stamen fusion: totally free (0); fused (1). 19. Nectar well: upper stamens not modified (0); upper stamens modified to form a circular opening (1). 20. Anthers: not dimorphic or slightly dimorphic (0); strongly dimorphic (1). 21. Ovule number: several ovules (0); 1 or 2 ovules (1). 22. Seed aril: non-fleshy (0); fleshy (1). 23. Seed aril shape: aril not extended towards the lens (0); aril extended towards the lens (1). 24. Seed aril: not interrupted at micropylar end (0); interrupted (1). 25. Micropyle shape: ypsaloid (0); punctate (1): 26. Piperidyl alkaloids: low concentrations or absent (0); present as major compounds in most taxa (1). 27. Tetracyclic quinolizidine alkaloids: present (0); absent (1). 28. Carboxylic acid esters: present (0); absent (1). 29. Ammodendrine: absent (0); present as a major compound in at least some taxa (1). Relationships in the Podalyrieae and Liparieae 21

LIPARIEAE PODALYRIEAE

'F. O~ ~ ._~ ~- >-Z ~- ~J

16 4

'26

:21: 24 20 ~: i t,11 (I 45 = 1210

: 21

:23 ez~:3-

Fig. 9. Fully resolved cladogram of relationships at tribal and generic level, based on Table 2. • apomorphy without homoplasy; [] apomorphy with subsequent reversal; = convergence; x reversal

Hypocalyptus (Table 2). Cadia was also included in the investigation to determine whether it should be included in the Podalyrieae. Information on the Crotalarieae came from VAN WYK & SCHtrrzE (1995b). Data for the Mirbelieae were taken from POLHrLL (1976, 1981C) and C~sP & WESTON (1987, 1995), whilst VAN DEr~ MAESEN (1970) was consulted for details on the genus Cadia. A single fully resolved 22 A.L. SCHUTTE& B.-E. VAN WYK:

I.g

i -~ E ,._ ...- ~ .- ," ~ E , , ~ .~ .~- s;

-~19 c19 ~14 ~14 ~10 c10 ~8 <8 :27 30 =33 )16 " 31 23 :6 "Z931 = 24 )16 33 1 ~9 ;.-M 20 <6 I0 .,., ,o 1 !!

,27 ,24 P 27 ,22 -23 c21 22

=31 =30

i20 26 ~18 ~19 .•,29 = 27 ~13 =20 ,2.•8:29 =19 ) 25 26 17 :10 22 .J_,: =8 b17 =19 P17 ,15 )15 =14 =14 ~:3 116 ,12 112 =8 =8

~32 I132 ~28 =23 =10 =21 2,1- =6 18

Fig. 10. Fully resolved cladograms of relationships in the Podalyrieae and Liparieae (excluding Hypocalyptus). Topology A is based on Table 3, with the Mirbelieae as outgroup. Topology B is based on Table 4, with the Crotalarieae as outgroup and topology C on Table 5, with Cadia as outgroup. - Symbols as in Fig. 9

cladogram was generated from the data set (Fig. 9; consistency index 61, length 44, retention index 77). The result clearly indicates that the Podalyrieae plus Liparieae are monophyletic, with at least two apomorphies supporting the clade. Furthermore, it is also evident that Hypocalyptus is quite unlike any of the other genera or tribes included in the investigation. Cadia does not seem to fit into the Podalyrieae either. In the remaining three analyses, the relationships between the genera of the Podalyrieae and Liparieae were examined, using the Mirbelieae, Crotalarieae and Cadia as outgroups respectively. The data sets are shown in Tables 3-5. Hypocalyptus was omitted from these investigations, since it dropped to a basal position in the previous analysis. Each analysis resulted in one fully resolved Relationships in the Podalyrieae and Liparieae 23 topology (Fig. 10 A-C), but with the Mirbelieae as outgroup, the cladogram had a consistency index of 70 (length 47, retention index 80), with the Crotalarieae as outgroup, a consistency index of 66 (length 48, retention index 78) and with Cadia as outgroup, a consistency index of 70 (length 41, retention index 80). Two major clades are apparent in all three the cladograms, each supported by at least three synapomorphies: (1) a Xiphotheca clade, with Amphithalea and Coelidium, and (2) a Podalyria clade, with Cyclopia, Liparia, Stirtonanthus, 1,7rgilia and Calpurnia. In the Xiphotheca clade, a sister relationship between Amphithalea and Coelidium is supported by three shared derived characters. The only obvious difference between topologies A and B, lies in the positions of Cyclopia and Liparia in the Podalyria clade, where Cyclopia is basal in cladogram A, and Liparia basal in cladogram B. In topology C, however, the sequence of genera in the Podalyria clade is completely the reverse of A and B.

Discussion Convincing evidence for an enlarged Podalyrieae (excluding Hypocalyptus) is provided by at least two synapomorphies (Fig. 9), namely the strongly reduced or absent bracteoles and the occurrence of persistent antipodals in the female gametophyte. To these may be added the accumulation of esters of anthocyanins in all the pink, purple and orange-flowered species. Other apomorphies include the absence of canavanine in the seed, punctate micropyle and interrupted seed aril, depending on which outgroup is used. Previously, VAN WYK & SCHUTTE (1995b) also found the two tribes to be monophyletic if Hypocalyptus is omitted, but both tribes were monophyletic on their own and it was therefore decided to retain them as separate taxa. In the cladograms presented above, however, Liparia grouped with the Podalyrieae clade rather than the Xiphotheca clade, thereby implicating a stronger relationship with the former than the latter. Based on the available data, the present topology appears to be an improved representation of possible relationships between the tribes. Liparia has more characters in common with the Podalyria clade, than with the Xiphotheca group. In fact, the basal split into these two phylogenetic groups resulted from each analysis, regardless of the choice of outgroup. The two main clades are given subtribal status in an enlarged Podalyrieae in the taxonomic treatment below. This provides sound evidence for earlier suggestions (POLHILL 1976, 1981d, e; VAN WYK & SCHUTT~ 1995b) that the Podalyrieae and Liparieae should be amalgamated. The primary reason for the separation between the two tribes has been weighting of the stamen character. Traditionally free stamens have been used as the essential feature of the Podalyrieae, but in recent years it has been recorded to segregate some natural groups. For instance, a diadelphous stamen arrangement has excluded Calpurnia from the Podalyrieae. VAN WYK SCHUTTZ (1995b) proved that this character is over-shadowed by at least four synapomorphies, indicating a strong sister relationship between Virgilia and Calpurnia. Moreover, it has been found that the stamen character breaks down in some genera, e.g. Podalyria, where they are often strictly diadelphous (SCHUTTE, pers. obs.). 24 A.L. SCHUTTE& B.-E. VAN WYK:

The three cladograms in Fig. 10 A-C are identical, except for variation in the sequence of branching in the Podalyria clade. Topologies A and B differ only in the positions of Liparia and Cyclopia. In topology A, Cyclopia is basal to the clade, with Liparia, Podalyria, Stirtonanthus, Virgilia and Calpurnia forming a monophyletic group, supported by two synapomorphies (i.e. presence of tetracyclic quinolizidine alkaloids and esterification of monohydroxylated lupanines). In this tree, Liparia is well-separated from the Xiphotheca clade and the uniqueness of Cyclopia seems to stand out. Topology B has Liparia basal to the clade, thereby reflecting its affinity with the Xiphotheca group. Apart from its strong relationship with the Podalyria group, Liparia also has a number of characters in common with Xiphotheca, Amphithalea and Coelidium (e.g. strongly reduced or absent petioles and accumulation of ammodendrine). In this clade, Cyclopia forms a natural group with Podalyria, Stirtonanthus, Virgilia and Calpurnia, which is subtended by two apomorphies (i.e. slightly fused or free stamens and absence of ammodendrine). The sequence of branching of the Podalyria clade in topology C is precisely the reverse of the other two topologies. From a biogeographical point of view, this sequence is probably the most reasonable hypothesis of evolutionary relationships, in terms of a progression from a widespread to a reduced distribution area. [Calpurnia is a widespread summer rainfall genus, occurring from the southern Cape region in South Africa, northwards along the highlands of Africa to India. All the other genera are restricted to the winter rainfall Cape Floristic Region of South Africa (ScmrrTE & VLOK, unpubl.).] Yet in this case, Cadia was used as outgroup, which in the first analysis proved not to be closely related to the Podalyrieae. The taxonomic significance of additional chemical and genetic characters is currently under investigation (i.e. electrophoretic, flavonoid and DNA studies). It is not impossible that these or other future studies may indicate that Cadia should be moved to a position closer to the Podalyrieae. According to the results, the Podalyrieae appear to be well-placed between the African Crotalarieae and the Australian clade. There is no valid evidence to support a direct connection between the Podalyrieae and the Australian tribes, as has been proposed by CRISP & WESTON (1987). However, little doubt exists about the affinity between the African Podalyrieae and Crotalarieae (VAN WYK & ScmrrTE 1995b). We therefore favour topology B, where the Crotalarieae were used as outgroup. In this cladogram, Liparia is basal to the Podalyria clade and not far removed from the Xiphotheca group, with which it also shares a number of characters. Hypocalyptus remains a problem as far as its taxonomic position is concerned. It evidently does not fit into any of the tribes included in this study. A paper dealing with its position and status within subfam. Papilionoideae will be published elsewhere (SCHUTTE & VAN WY~: 1997).

Taxonomic treatment Tribe Podalyrieae BENTH. emend. A. L. SCHUTTE emend, nov., BENTH. Comm. Leg. Gen., 1 (1837); BENTH. in Ann. Wien. Mus. 2:65 (1839); HARV. in HARV. & SOND., F1. Cap. 2:2 (1862); HUTCH., Gen. F1. P1. 1:336 (1964); POLHmL in POLHILL & RAVEN, Adv. Leg. Syst. 1:396 (1981); YAKOVLEV,Bobovye Zemnogo Relationships in the Podalyrieae and Liparieae 25

Shara, 85 (1991); VAN WYK & SCHUTTE in CPdsP & DOYLE, Adv. Leg. Syst. 7:304 (1995). Loteae DC. subtribe Lipariinae BENTH. in Hoo~:., London J. Bot. 2:441 (1843), as ' Liparieae' . Genisteae BEYrI4. subtribe Lipariinae (BENTH.) BEYrI-I. in BENa'H. & HOOK. f., Gen. P1. 1:439 (1865), as 'Liparieae'. Liparieae (BENTH.) HARV. in HARV. & SOND., F1. Cap. 2:2 (1862) syn. nov.; HuTcH., Gen. F1. P1. 1:346 (1964); PoI~HIII in Bot. Syst. 1:313 (1976) p. p.; POLHmL in POLHmL & RAVEN, Adv. Leg. Syst. 1:398 (1981) p. p.; VAN WYK & SCHUTTE in CRISP & DOYLE, Adv. Leg. Syst. 7:304 (1995). Liparieae (BENTH.) HARV. subtribe Lipariinae (BENTH.) BENTH., YAKOVLEV, Bobovye Zemnogo Shara, 87 (1991) syn. nov. Trees, shrubs or subshrubs. Leaves imparipinnate, digitately trifoliolate or simple; petiole present or reduced to a pulvinus only; leaf bases + prominent; stipules present, sometimes strongly reduced. Inflorescences axillary racemes or terminal panicles, 1-several -flowered; peduncles present or absent; bracts small or enlarged, often sheathing at the base, sometimes brightly coloured; bracteoles rarely present. Pedicels present or absent. Calyx with base intrusive or with a prominent hypanthium; two upper lobes fused higher up than lower three; carinal lobe as large as or sometimes much larger than the others. Corolla yellow, pink, mauve, white or reddish-orange, totally glabrous. Bracteoles strongly reduced or absent. Standard thickly textured with strongly reflexed claw or thinly textured with slightly reflexed claw; base sometimes auriculate; apex emarginate. Wing petals sometimes auriculate and pocketed; petal sculpturing sometimes present. Keel petals obtuse, shortly or strongly beaked, sometimes auriculate; pocket present or absent. Stamens 10, free, monadelphous or diadelphous; anthers subequal to dimorphic, alternately short dorsifixed, long basifixed or subbasifixed. Pistil sessile; ovary 1-22 -ovuled; style curved upwards with base often hairy. Pods coriaceous, dehiscent, mostly inflated, sometimes compressed between the seeds. Seeds reniform, with a small radicular lobe; micropyle punctate; aril fleshy collar-like or non-fleshy rim. Chromosome number 2n = 18, 36, 54, -t-126. Nine genera, all restricted to the Cape, except Calpurnia which extends through Africa to India.

Key to the genera 1. Leaves imparipinnate, leaflets several ...... 2 1. Leaves digitate or simple, leaflets 1 or 3 ...... 3 2. Flowers pink, purple or white; stamens free ...... 8. Virgilia 2. Flowers yellow; stamens fused ...... 9. Calpurnia 3. Leaves digitately 3-foliolate; bracts paired ...... 5. Cyclopia 3. Leaves simple; bracts single ...... 4 4. Stamens free almost to the base ...... 5 4. Stamens diadelphous or monadelphous ...... 6 5. Flowers yellow, decussate (arranged in opposite pairs of 2, 4 or 6 flowers); seeds with a non-fleshy rim-aril ...... 7. Stirtonanthus 26 A.L. SCr~UTTE& B.-E. VAN WYK:

5. Flowers pink, mauve or white, racemose (1-5 flowers per raceme); seeds with a fleshy collar-like aril ...... 6. Podalyria 6. Calyx with base intrusive; carinal lobe of calyx usually longer than the upper 4 lobes; leaves sessile, 3- or more-veined from the base; inflorescences 4- many- flowered (rarely 2-flowered); bracts often leaf-like ...... 4. Liparia 6. Calyx with base gradually narrowing to the pedicel; carinal lobe of calyx usually as long as the upper 4 lobes; leaves usually petiolate or at least with a pulvinus, single veined from the base; inflorescences 1- or 2-flowered; bracts not leaf-like ...... 7 7. Flowers yellow, fading to brown with age; bracteoles often present; aril not extended towards the lens; pods compressed between the seeds... 1. Xiphotheca 7. Flowers mostly pink, mauve or white (if rarely yellow, then less than 10 mm long and not fading to brown with age); bracteoles totally absent; aril extended towards the lens; pods not compressed between the seeds ...... 8 8. Vexillary filament fused with other filaments ...... 3. Coelidium 8. Vexillary filament free down to hypanthium ...... 2. Amphithalea

Subtribe Xiphothecinae A. L. SCHUTTE subtribus nova; subtribus Podalyr- iinis affinis, a qua basis calycis non intrusis et apicibus carinis obtusis differt. Type: Xiphotheca ECr~L. & ZEYI-I. This subtribe is recognized by its non-intrusive calyx base, obtuse keel apex, reduced number of ovules and the wing petals, which have a thickened lobe on the abaxial surface. Three genera constitute the subtribe. 1. Xiphotheca Ec~. & ZEYH., Enum. P1. Afric. Austral. 2:166 (1836); ScnvxxE & VAN WYK in Taxon 42:45 (1993); VAN WYK & SCr~UTTEin Cease & DOYLE, Adv. Leg. Syst. 7:305 (1995); ScI-Itrrx~ in Ann. Missouri Bot. Gard. 84:93 (1997). Nine species. 2. Amphithalea Ecrd~. & ZEYI-I.,Enum. 2:167 (1836); BENTrI. in HooK., London J. Bot. 2:442 & 449 (1843); HARV. in HARV. & SOND., F1. Cap. 2:21 (1862); Htrrca., Gen. F1. P1. 1:347 (1964); DYER, Gen. 1:247 (1975); POLI~ILLin Bot. Syst. 1:316 (1976); POH-IILL in POLHV-L & RAVEN, Adv. Leg. Syst. 1:398 (1981); GRax',q3Y in Opera Bot. 80:12 (1985); VAN WYK & SCI-IUTTEin CRISP & DOYLE, Adv. Leg. Syst. 7:305 (1995). Twenty-one species. 3. Coelidium VOGEL ex WALP. in Linnaea 13:472 (1839).; BENTI-I. in HooI,:., London J. Bot. 2:442 & 453 (1843); HARV. in HARV. & SOND., F1. Cap. 2:24 (1862); Htrrc~I., Gen. F1. P1. 1:350 (1964); DYER, Gen. 1:248 (1975); POLHILLin Bot. Syst. 1:316 (1976); Grub',mY in Opera Bot. 54:12 (1980); POLHILLin POI~IaILL& RAVEN, Adv. Leg. Syst. 1:398 (1981); VAN WYK & ScrarrxE in CRISP & DOYLE, Adv. Leg. Syst. 7:305 (1995). Twenty-one species.

Subtribe Podalyriinae Subtribe Podalyriinae has an intrusive calyx base and a beaked keel tip. It consists of six genera. 4. Liparia L., Mant. 2:156 (1771); DC., Prodr. 2:121 (1825); BBNTI-I.in HooK., London J. Bot. 2:442 (1843); HARV. in HARV. & SOND., F1. Cap. 2:14 (1862); HuTcH., Gen. F1. P1. 1:347 (1964); Bos in J. S. Afr. Bot. 33:272 (1967); DYER, Gen. 1:246 (1975); POLIamL in Bot. Syst. 1:315 (1976); POLI4ILL in POLH~L & Relationships in the Podalyrieae and Liparieae 27

RAVEN, Adv. Leg. Syst. 1:397 (1981); SCHU~E & VAN WYK in Taxon 43:577 (1994); VAN WYK & SCHt~TTE in CRISp & DOYLE, Adv. Leg. Syst. 7:305 (1995); ScntrrxE in Nordic J. Bot. 17 (1): (1997c). Twenty species. 5. Cyclopia VEN~r., Dec. Gen. Nov., 8 (1808); DC., Prodr. 2:101 (1825), M6m. L6g., 167 (1826); BENTH. in Ann. Wien. Mus. 2:67 (1839); BEWrH. in HOOK., London J. Bot. 2:432 (1843); HARV. in HARV. & SOND., F1. Cap. 2:6 (1862); HO~WrEYR & E. PHrLLIPS in Bothalia 1:106 (1922); KIES in Bothalia 6:162 (1951); HUTCH., Gen. F1. P1. 1:343 (1964); DYER, Gen. 1:246 (1975); POLHmLin POLHILL& RAVEN, Adv. Leg. Syst. 1:397 (1981); VAN WYK & SCm~TTEin CRISP & DOYLE, Adv. Leg. Syst. 7:304 (1995); SCHtrrTE in Edinburgh J. Bot. 54 (2): (1997d). 23 species. 6, Podalyria LAM., Illustr. 2: 454, t. 327, f. 3 (1793); DC., Prodr. 2:101 (1825); Eel. & ZEVH., Enum. 2:155 (1836); E. MEY., Comm. 1:4 (1836); BENTH. in Ann. Wien. Mus. 2:67 (1839); BE~CrH. in Hook., London J. Bot. 2:435 (1843); HARV. in HARV. & SOND., F1. Cap. 2:9 (1862); HUTCH., Gen. F1. P1. 1:339 (1964); DYER, Gen. 1:246 (1975); POLHn~L in POLHILL & RAVEN, Adv. Leg. Syst. 1:397 (1981); VAN WYK & ScmrrrE in CRIsp & DOYLE, Adv. Leg. Syst. 7:304 (1995). Nineteen species. 7. Stirtonanthus B.-E. VAN WYK & A. L. ScmrrTE in Nordic J. Bot. 15:67 (1995). Three species. 8. Virgilia Pore., Encycl. 8:677 (1808); DC., Prodr. 2:98 (1825); E. MEY., Comm. 1:1 (1836); HARV. in HARV. & SOND., F1. Cap. 2: 2:266 (1862); HUTCH., Gen. F1. P1. 1:323 (1964); YAKOVLEVin Bot. Zhurn. 55:837 (1970); PALMER & PITMAN, Trees S. Aft. 2:903 (1973); DYER, Gen. 1:245 (1975); COATES PALGRAVE, Trees. S. Aft., 301 (1977); POLHn~L in POLHILL & RAVEN, Adv. Leg. Syst. 1:397 (1981); VAN WYK in S. African J. Bot. 52:348 (1986); VAN WYK & SCmTTTE in CRISP & DOYLE, Adv. Leg. Syst. 7:304 (1995). Two species. 9. Calpurnia E. MEY, Comm. 1:2 (1836); BENTH. in Ann. Wien. Mus. 2:89 (1839); HARV. in HARV. & Sow., F1. Cap. 2:266 (1862); HtrrcH., Gen. F1. P1.1:329 (1964); DYER, Gen. 1:244 (1975); POL~ILL in POLHmL & RAVEN, Adv. Leg. Syst. 1: 229 (1981); VAN WYK & SCHtrrTE in CRISP & DO~E, Adv. Leg. Syst. 7:304 (1995). Seven species.

This study formed part of the first author's Ph.D. Thesis in Botany at the Rand Afrikaans University. We thank Drs G. H. VERDOORN,R. GREINWALDand R BACHMANNfor their contributions to the alkaloid research project and Mr R J. D. W~TER for his involvement with the flavonoid study. Financial support from the Rand Afrikaans University and Foundation for Research Development is acknowledged.

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Addresses of the authors: ANNE LISE SCHUTTE (present address: Compton Herbarium, Kirstenbosch, National Botanical Institute, Private Bag X7, 7735 Claremont, South Africa) B.-E. VAN WYK, Department of Botany, Rand Afrikaans University, R O. Box 524, 2006 Auckland Park, Johannesburg, South Africa.

Accepted December 11, 1996 by E EHRENDORFER